Techniques of tissue engineering employing biocompatible scaffolds provide viable alternatives to prosthetic materials currently used in prosthetic and reconstructive surgery (e.g., craniomaxillofacial and spinal surgery). These materials also hold promise in the formation of tissue or organ equivalents to replace diseased, defective, or injured tissues. Compatible, biodegradable materials may be used for scaffolds which initiate and sustain tissue or bone growth, but which are naturally degraded over time within the body. Such materials may also be used for controlled release of therapeutic materials (e.g., genetic material, cells, hormones, drugs, or pro-drugs) into a predetermined area. Polymers, such as polylactic acid, polyorthoesters, and polyanhydrides, used to create these scaffolds are difficult to mold and, result in, among other things, poor cell attachment and poor integration into the site where the tissue engineered material is utilized. With some exceptions, they also lack biologically relevant signals.
Self-assembled peptide-amphiphile nanofibers have been used to direct the growth of biominerals such as hydroxyapatite. These nanofibers are comprised peptide-amphiphiles, that are comprised of a hydrophobic aliphatic tail coupled to a relatively hydrophilic peptide head group. The peptide head group may include at least two segments: a structural segment and a functional segment. Structural segments may include between 2 and 4 cysteine residues may be used to covalently stabilize the self-assembled peptide amphiphile structures via disulfide bond formation between individual peptide amphiphile molecules within a fiber. Alternatively, the structural segment may contain other residues, such as serine, leucine, alanine, or glycine for example. Though these residues may not promote covalent stabilization of the nanofibers, they may participate in structural organization, such as beta-sheet formation, in the assembled nanofibers. The functional head group may be composed of different amino acid combinations and include moieties such as carboxyl, thiol, amine, phosphate, and hydroxyl functional groups located near the end of the molecule most distant from the molecule's aliphatic tail. Examples of carboxyl group-containing residues include aspartic acid or glutamic acid. Examples of amine or guanidinium-containing residues include lysine or arginine respectively. When the peptide amphiphiles are self-assembled under aqueous conditions, it is expected that these functional residues will be displayed near the self-assembled micelle (generally a nanofiber) surface where they may be available for reaction with other moieties to bind the peptide amphiphile.
The versatility and functionality of these self-assembling nanofibrous materials may prove to be useful in tissue repair, cell growth, or organ reconstruction. The term tissue includes muscle, nerve, vascular, and bone tissue and other common understandings of tissue. The present invention may also find application in regulation, inhibition or promotion of axon outgrowth in neurons as well as the regulation, inhibition or promotion of cell-substrate adhesion among nerve cells. Coating these peptide amphiphile compositions on surfaces of scaffolds and implants, for example stainless steel stents, electrodes for electrical stimulation of nerves, or metal-based orthopedic implants, may furthermore enhance existing tissue engineering strategies. Importantly, multiple peptide signals may be used in the same supramolecular self assembled peptide amphiphile to accomplish different and potentially synergistic effects.
The peptide amphiphile composition(s) of such a system may include a peptide component having residues capable of intermolecular cross-linking. The thiol moieties of cysteine residues can be used for intermolecular disulfide bond formation through introduction of a suitable oxidizing agent or under physiological conditions. Conversely such bonds can be cleaved by a reducing agent introduced into the system or under reducing conditions. The concentration of cysteine residues, when utilized, can also be varied to control the chemical and/or biological stability of the nanofibrous system and therefore control the rate of therapeutic delivery or release of cells or other beneficial agent, using the nanofibers as the carriers. For example, enzymes could be incorporated into such nanofibers to control their biodegradation rate through hydrolysis of the disulfide bonds. Such degradation and/or the concentration of the cysteine residues can be utilized in a variety of tissue engineering applications. The thiol functionality of such peptide amphiphiles may also be useful for binding the supramolecular structures to surfaces.
The complimentary nature of the biological portions of the peptide amphiphiles may mimic amino acid sequences found in naturally occurring peptides. Self-assembled gels composed of peptide-amphiphile nanofibers with the RGD peptide sequence mimic the function of collagen fibrils to organize and direct the growth of the hydroxyapatite crystals. Other potentially useful amino acid sequences in such peptides may include the SEQ ID NO:1 YIGSR and SEQ ID NO:2 IKVAV amino acid sequences. Such amino acid sequences in self assembled peptide amphiphiles may have a synergistic effect on cell growth and nerve regeneration. The growth of cells on substrates implanted or delivered to the body would be beneficial to implantation of artificial hearts, restoring nerve function, healing of grafting blood vessels; forming skin grafts and preparing “artificial skin” by culturing epidermal cells on a fibrous laffice.
Damage to the endothelial and medial layers of a blood vessel, such as often occurs in the course of balloon angioplasty and stent procedures, has been found to stimulate neointimal proliferation, leading to restenosis of atherosclerotic vessels. The normal endothelium, which lines blood vessels, is uniquely and completely compatible with blood. Endothelial cells initiate metabolic processes which actively discourage platelet deposition and thrombus formation in vessel walls. Damaged arterial surfaces within the vascular system are highly susceptible to thrombus formation. While systemic drugs have been used to prevent coagulation and to inhibit platelet aggregation, a need exists to treat the damaged arterial surface directly to prevent thrombus formation and subsequent intimal smooth muscle cell proliferation.
Stents made up of metals such as titanium and its alloys have been designed to promote organized endothelial cell growth. Such stents comprise a plurality of depressions in the surface of at least a portion of the stent body, preferably arranged in a regular pattern on at least the interior surface of the stent body, such as a waffle weave. Other stents have surface features which comprise a plurality of pleats, ridges, channels or pores in the stent body wherein at least some of the pores run between the interior and exterior sides of the stent body (i.e., penetrate the stent body) and are sized to promote the organized cell growth.
The directed growth of cells, for example nerve cells and endothelial cells, on implantable surfaces and scaffolds would be desirable for the regeneration and growth of cells, organs, and tissue within the body. It would be desirable to provide surgical implants that may facilitate the growth of tissue, vascular tissues, nerve, and cells on or in tissue surrounding the surgical implant. It would be desirable for new and better scaffolds, implants, stents and electrode for placement into a body that are adapted to promote growth of infiltrating cells into organized cellular structures, such as take place during angiogenesis and/or neovascularization, to aid in repair of damaged body organs and vessels.
As part of a related consideration, titanium and its alloys have been used extensively as skeletal implant materials where the metals' high strength to weight ratio, toughness and the bioinert character of the naturally forming oxide layer have lead to widespread clinical success. As tissue engineering has developed, however, researchers have explored the use of calcium phosphate coatings on titanium-based implant surfaces to introduce an element of bioactivity to the otherwise inert oxidized metal surfaces. In vitro studies have shown that calcium phosphates may form osteoconductive coatings which enhance cellular attachment and proliferation. In vivo models have shown an improvement in implant interfacial strength when titanium surfaces are coated with various calcium phosphate coatings, often hydroxyapatite (Ca10(PO4)2(OH)2). Studies have also shown that degradation of these calcium phosphate coatings at implant-tissue interfaces facilitates the accelerated formation of de novo bone.
Commonly used methods for coating Ti with these calcium phosphate coatings include plasma spraying, electrophoresis, sol-gel, and solution-phase precipitation. Methods such as plasma spraying or sol gel tend to produce dense, often highly crystalline apatitic phases with little or no phase selectivity, and some of these methods are also unable to coat interior surfaces of porous titanium structures. Many of these methods for growth involve extremely long growth times, weeks to months, offer little control over crystal size or shape, and lack any added chemical functionality, such as that afforded by organic macromolecules. Organic macromolecules have been known to play roles in biomineral crystal modification. Additionally, where clusters form on porous surfaces, surface coating is frequently less than 100%. Solution-phase growth, however, enables nucleation of calcium phosphate coatings directly on implant surfaces, even porous surfaces. In addition, this wet chemical approach allows for the formation of not only hydroxyapatite, but also other biologically relevant calcium phosphate phases, such as octacalcium phosphate, (Ca8H2(PO4)6.5H2O), a precursor to hydroxyapatite. Solution-phase growth of these coatings also allows for the introduction of organic macromolecules into the coating, a feature not possible with some of the high temperature coating processes, such as plasma spraying.
Work has been done investigating the interactions of various biological macromolecules with calcium phosphate coatings. The growth of calcium phosphate coatings in the presence of biomolecules such as albumin, fibronectin, and poly(aminoacids), is substantially inhibited. Poly(L-lysine), for example, is a well-established cell adhesion promoter with excellent chemical functionality, but has been shown to inhibit apatite growth on a titanium alloy surface. Poly(amino acids) have been used as nucleating agents and macromolecular tethers to address this problem by growing poly(L-lysine)-containing organoapatite onto poly(amino-acid)-coated titanium-based surfaces. This method uses poly(amino acids) in several of the coating steps and layers; it also produces relatively bulky clusters of organoapatite, which may be disadvantageous in coating structures with fine porous textures. An alternative approach investigated is growing a calcium phosphate coating containing albumin onto a preexisting calcium phosphate layer.
It would be desirable to form polyamine-modified nanotextured calcium phosphate coating on implantable metal surfaces. Grown onto calcium phosphate seeds the new material combines the versatility and simplicity of solution-phase calcium phosphate growth on an implantable surface with the chemical and biological functionality of a poly(amine).
It would be desirable to coat the surfaces of materials with biominerals so that substantially all of the surface is coated, and that the coating provides a favorable surface for chemical modification, attachment of peptide amphiphile nanofibers, cell and tissue growth and adhesion. It would further be desirable if the coating could be applied to a material suitable for implant into a patient and that the coating be degradable under physiological conditions.